Listeria monocytogenes is a facultative, intra-cytoplasmic bacterial pathogen that is responsible for serious infections in immunocompromised patients, pregnant women, and neonates. L. monocytogenes infections are primarily food-borne and listeriosis is the leading cause of death from food-related illness. In addition to its significance as a human pathogen, L. monocytogenes also serves as a useful model system for exploring the intracellular interactions that take place between parasite and host. L. monocytogenes is capable of sensing the different host cell compartment environments it encounters during the course of infection and of responding with the regulated expression of virulence factors. The PrfA protein of L. monocytogenes is a key transcriptional activator of virulence gene expression. PrfA contributes to the temporal regulation of L. monocytogenes gene expression within host cells, but the mechanisms used by the bacterium to coordinate intracellular gene expression are unknown. The goal of these studies is to elucidate the mechanisms that govern intracellular gene expression and to identify L. monocytogenes gene products that are subject to this intracellular regulatory network. In Aim 1, experiments are designed to analyze the mutations in L. monocytogenes regulatory mutants with altered patterns of intracellular gene expression and to define the effects of these mutations on virulence gene regulation. These studies should lead to the identification of new components that may act in concert with PrfA and that contribute to intracellular regulation of gene expression. Aim 2 will define functional regions of PrfA that promote activation of virulence gene expression. The contributions of specific functional domains of PrfA to virulence gene regulation in L. monocytogenes will be assessed. In Aim 3, studies are designed to identify additional L. monocytogenes gene products whose expression or activity is PrfA-dependent, and assess the contributions of these products to bacterial pathogenesis. The proposed studies should further our understanding as to how an intracellular bacterium senses the environment of different host cell compartments and regulates expression and activity of its virulence factors in response. This information is important towards better definition of the interactions that occur between host and pathogen during the process of infection.